cDNA Library Preparation Research Articles

Generation and analysis of a mouse multitissue genome annotation atlas.

Generating an accurate and complete genome annotation for an organism is complex because the cells within each tissue can express a unique set of transcript isoforms from a unique set of genes. A comprehensive genome annotation should contain information on what tissues express what transcript isoforms at what level. This tissue-level isoform information can then inform a wide range of research questions as well as experiment designs. Long-read sequencing technology combined with advanced full-length cDNA library preparation methods has now achieved throughput and accuracy where generating these types of annotations is achievable. Here, we show this by generating a genome annotation of the mouse (Mus musculus). We used the nanopore-based R2C2 long-read sequencing method to generate 64 million highly accurate full-length cDNA consensus reads-averaging 5.4 million reads per tissue for a dozen tissues. Using the Mandalorion tool, we processed these reads to generate the Tissue-level Atlas of Mouse Isoforms which is available as a trackhub for the UCSC Genome Browser and contains at least one full-length isoform for the vast majority of expressed genes in each tissue.

CapTrap-seq: a platform-agnostic and quantitative approach for high-fidelity full-length RNA sequencing

Long-read RNA sequencing is essential to produce accurate and exhaustive annotation of eukaryotic genomes. Despite advancements in throughput and accuracy, achieving reliable end-to-end identification of RNA transcripts remains a challenge for long-read sequencing methods. To address this limitation, we develop CapTrap-seq, a cDNA library preparation method, which combines the Cap-trapping strategy with oligo(dT) priming to detect 5’ capped, full-length transcripts. In our study, we evaluate the performance of CapTrap-seq alongside other widely used RNA-seq library preparation protocols in human and mouse tissues, employing both ONT and PacBio sequencing technologies. To explore the quantitative capabilities of CapTrap-seq and its accuracy in reconstructing full-length RNA molecules, we implement a capping strategy for synthetic RNA spike-in sequences that mimics the natural 5’cap formation. Our benchmarks, incorporating the Long-read RNA-seq Genome Annotation Assessment Project (LRGASP) data, demonstrate that CapTrap-seq is a competitive, platform-agnostic RNA library preparation method for generating full-length transcript sequences.

Identification of Early Transcriptional Markers of Autosomal Dominant Polycystic Kidney Disease Cystic Epithelial Cells

Study objective: Mechanisms driving autosomal dominant polycystic kidney disease (ADPKD) cystogenesis, a hallmark of this lethal monogenic disease, are not well understood and reliable biomarkers are scarce. We sought to explore transcriptional changes in ADPKD models with suppressed and non-suppressed cystic phenotype to explore mechanisms driving ADPKD cystogenesis and identify potential biomarkers in disease progression. Hypothesis: Early-stage kidneys from a model in which cystic disease is suppressed by expression of the polycystin-1 C-terminal tail (CTT), a 200 amino acid portion of the larger polycystin-1 protein that is dysregulated in ADPKD, exhibit a differential transcriptional profile from early-stage kidneys in a non-suppressed model. Methodology: Orthologous doxycycline-inducible Pkd1fl/fl;Pax8rtTA;Teto-Cre ADPKD C57BL/6N mouse models expressing CTT ( Pkd1-KO+CTT) or not ( Pkd1-KO) were induced between 4-6 weeks and aged to 10 weeks. We performed single cell RNA sequencing (scRNA-seq) with single cell kidney suspensions from biological replicates that were pooled and processed for cDNA synthesis and library preparation with 10X Chromium technology. Reads were aligned using the 10X Genomics CellRanger pipeline and further processed using the gene cell-inverse cell frequency (gf-icf) pipeline. Data: Clusters corresponding to tubule segments were identified using established annotations. Several novel clusters defined by expression of multiple markers, including Jarid2, Tmtc2, and Filip1l, were identified in the Pkd1-KO model. These clusters were sparsely populated in Pkd1-KO+CTT samples, suggesting that they may correspond to cyst epithelial cells that are suppressed by CTT expression. Each of these novel clusters included cells whose markers identified them from distinct tubule segments (e.g. Sglt2, Umod, S100g), suggesting that cells of different segments are clustered together by virtue of sharing the putative cyst cell transcriptional signature. Importantly, at 10 weeks there are no significant differences in disease endpoints such as kidney weight to body weight, serum creatinine, blood urea nitrogen, or kidney morphology between the Pkd1-KO+CTT and Pkd1-KO models, suggesting that the cells assigned to the cystic epithelial cell clusters exhibit early identifiable differences in transcriptional programming. Summary of results: scRNA-seq at an early timepoint reveals novel transcriptional makers of putative cystic epithelial cells. The protein expression levels of individual markers are being assessed. Conclusions: These results suggest that there are epithelial cells early in disease progression exhibiting a unique transcriptional signature that may elucidate the mechanisms of cyst formation and that may provide new candidate biomarkers. This work was funded in part by the NIH F31 DK135356 (VR), NIH RC2 DK120534-01 (MJC), PR191158 Department of Defense (MJC), and PKD 905682 Foundation for Research in Polycystic Kidney Disease Research Grant (MJC). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

First Report of Hibiscus Chlorotic Ringspot Virus infecting Hibiscus rosa-sinensis in Italy.

Hibiscus chlorotic ringspot virus (HCRSV) belongs to the Betacarmovirus genus of the Tombusviridae family and is a positive-sense monopartite single-stranded RNA virus. HCRSV was first described in a hibiscus variety imported into the United States from El Salvador. The HCRSV natural host range is limited to species belonging to Malvaceae family, in particular Hibiscus rosa-sinensis, H. manihot, H. diversifolius and H. syriacus. In September 2023, leaf mottle and chlorotic spots were observed on the leaves of two H. rosa-sinensis plants located in a public garden of Ercolano (Naples city, Southern Italy). Two leaves, one from each symptomatic plant, were pooled and submitted to double-stranded RNA extraction using a Viral Gene-Spin Viral DNA/RNA Extraction Kit (iNtRON, Korea), followed by cDNA library preparation with the TruSeq Stranded Total RNA (Illumina, USA). Sequencing on the Illumina NovaSeq 6000 platform (Illumina, USA) with 150-bp paired-end reads yielded 22,578,913 raw reads. 21,413,571 clean reads were obtained by quality control on the sequencing data was performed with the software FastQC (v. 0.11.5). Then low quality bases and adapters were removed with the software BBDuk in the BBTools package setting a minimum read quality of 25 and minimum read length of 35 bp. The resulted filtered reads were used to assembly viral genome by using two different algorithms (Metaspades and RNAViral) implemented in SPAdes (v. 3.15.3). Of the total contigs de novo assembled, the two algorithms implemented identified respectively 340 and 559 contigs related to viruses. BLASTn analysis of the contigs identify one coting of 3965 nt covering 97% to 100% of the whole genome sequence of nine HCRSV isolates, with percentage of identity of 87.8-95.2%. No other plant viruses or viroids have been identified during bioinformatic analysis. To confirm the result, a full-length genomic sequence of the Italian HCRSV isolate (named Ita-1), was obtained by reverse transcription polymerase chain reaction (RT-PCR) using specific primers designed on the sequence of the assembled contigs. The PCR products were sequenced using the Sanger method (Microsynth Seqlab, Germany) in both directions. The obtained full-length genomic sequence of the HCRSV isolate (Acc. No. OR981792) was 3910 nt in length. Maximum-likelihood phylogenetic trees inferred from the whole genome sequence showed that Ita-1 clustered closely with HCRSV isolates. The leaf samples were further analyzed using a HCRSV ELISA kit (Agdia, USA). Healthy H. rosa-sinensis leaves were taken as a negative control and buffer solution as a blank control. The results showed a positive reaction for the two symptomatic plants (OD = 1.345 ± 0.010 at 405 nm) relative to the negative (OD = 0.097) and blank (OD = 0.065) controls. Overall, the results of serological and molecular analyses supported that symptoms observed in H. rosa-sinensis were strictly associated to HCRSV infection. Four viruses have been reported in H. rosa-sinensis in Italy so far: eggplant mottled dwarf nucleorhabdovirus (EMDV), hibiscus latent Fort Pierce virus (HLFPV), hibiscus latent ringspot virus (HLRSV), hibiscus latent Singapore virus (HLSV). To our knowledge, this is the first report of HCRSV in H. rosa-sinensis in Italy, where probably infected plants were accidentally introduced. At present, HCRSV has been reported in three continents (Asia, Oceania and North America) and the number of countries where the virus has been detected is likely to increase rapidly as a result of increased surveillance and availability of diagnostic methods. This study will help the management of viral diseases on H. rosa-sinensis in Italy.

Sequencing: 10X Genomics 3' HT Assay for Gene Expression.

Single-cell RNA sequencing supports the isolation of individual cells and barcoding of cDNA, specific to each cell of origin. Subsequent sequencing of the generated library yields both the gene expression sequences and the cellular barcode, allowing distinction of gene expression patterns across individual cells. The 10X Genomics 3' HT assay uses a droplet-based method to isolate individual cells within oil emulsions, combined with a gel bead coated in uniquely barcoded primers, specific to each bead. The high-throughput, HT, assay is similar to its predecessor (3' v3.1) in reaction chemistry but utilizes (a) higher numbers of cellular barcodes, (b) a new, proprietary chip designed to target up to 60,000 cells per lane, and (c) captures up to 16 samples per run. The 3' HT assay supports whole cells and nuclei as input, with an approximate 60% capture rate. Here we describe the methods for sample quality control (QC) assays, loading and operation of the Chromium X instrument for cell capture, and cDNA synthesis and library preparation for downstream Illumina sequencing.

Discovery and Identification of Viruses Infecting Oomycetes.

This chapter describes protocols suitable for the detection and identification of RNA viruses infecting oomycetes (so-called water molds of Kingdom Heterokonta, Stramenopila), focusing on species of Phytophthora and exemplified by P. fragariae. The protocol includes laboratory procedures for oomycete cultivation and total RNA extraction from harvested mycelia, followed by instructions on suitable parameters given for sequencing companies on ribosomal RNA depletion, cDNA library preparation, and total RNA-sequencing (RNA-Seq). We also describe the bioinformatics steps needed for de novo assembly of raw reads into contigs, removal of host-associated contigs, and virus identification by database searches, as well as host validation by RT-PCR. All steps are described using an exemplar RNA-Seq library containing a yet undescribed fusagravirus hosted by a P. fragariae isolate.

Keloids are transcriptionally distinct from normal and hypertrophic scars.

Wound healing and skin regeneration after injury are complex biological processes, and deep injuries with a high degree of tissue destruction may result in severe scar formation. Clinically, scars can be classified into normal, hypertrophic and keloid scars. However, the molecular signature of each scar type is currently not known. The aim of this study was to reveal the transcriptional landscape of normal, hypertrophic and keloid skin scars following hand and plastic surgery based on total RNA sequencing. Eighteen skin scar samples from hand and plastic surgeries of human donors were minced directly after removal and stored in TRIzol (Thermo Fisher, USA). Samples were then subjected to RNA isolation, cDNA library preparation, bulk RNA sequencing and bioinformatics analysis. We show that keloid scars transcriptionally differed from normal and hypertrophic scars. Normal and hypertrophic scars presented overlapping clustering, and eight genes were shown to be commonly expressed between hypertrophic and normal scars. No genes were specifically expressed at a higher level in keloid and normal scars. Based on gene ontology pathway analysis, genes with a higher level of expression in keloid scars lead to increased (extra-) cellular matrix proliferation and cell interaction. Moreover, tumour-like genes were more highly expressed in keloid scars, supporting the clinical impression of strong and diffuse growth. This study furthers our understanding of the classification of differential scar types based on molecular signature, which may shed light on new diagnostic and therapeutic strategies for keloid scars in the future.

SDC2 Expression Is Increased in Myeloma Cells in Response to Loss of Pro-Survival Surface Proteins, CD28 and CD86

Introduction: While recent advances in multiple myeloma (MM) have improved overall survival, most patients will succumb to late-stage and drug resistant disease. Plasma cell (PC) survival outside of the bone marrow is characteristic of late-stage myeloma and pivotal to disease progression. PCs are typically restricted to the bone marrow microenvironment, as they are dependent on stromal interactions for survival. One such interaction is the engagement of CD28 and its ligand CD86, proteins which are best understood for their roles in T-cell activation by antigen presenting cells. However, myeloma cells can express both CD28 and CD86 on the cell surface. Notably, high CD28 expression is seen in 59% of medullary recurrences, and over 90% of extramedullary relapses. Moreover, high CD86 expression is more common in malignant plasma cells as compared to precursor stages and has a reported role in driving expression of IRF4. We have previously shown that expression of CD28 and CD86 is associated with worse outcomes than that observed in patients with low expression in the MMRF CoMMpass study (NCT01454297). We have also shown that silencing of CD28 or CD86 results in cell death and loss of IRF4 protein expression in human myeloma cell lines (HMCL). We confirmed these studies using CRISPR/Cas9 editing and demonstrated loss of proliferation and induction of cell death in HMCLs. However, the mechanism(s) by which CD28 and CD86 contribute to MM cell survival is not fully understood. Methods: To shed light on the mechanistic basis of CD28 and CD86 in the survival of MM, we used CRISPR/Cas9 editing to disrupt expression from RPMI8226 myeloma cells. We then passaged transduced cells to select for populations that can survive loss of CD28 or CD86. These passaged cells were sorted into populations that either retained CD28 or CD86 expression as transduction controls (High), or populations that underwent CRISPR/Cas9 depletion (Low). Surface expression of CD28 High/Low and CD86 High/Low populations was confirmed via flow cytometry and were then processed for RNA isolation and cDNA library preparation. RNA-sequencing was performed using paired-end sequencing, and data were aligned using the STAR aligner. Differentially expressed genes were determined using edgeR with an FDR ≤ 0.05 and a fold-change ≥ 2.0. Validation of RNA-sequencing results was further assessed via western blot and flow cytometry. Results: Analysis of RNA-sequencing results indicated that 57 genes were differentially expressed between High and Low cells, with 23 genes common to both CD28 and CD86 High/Low comparisons. (FDR<0.05). Notably, our results reveal that loss of CD28 or CD86 is associated with an increase in mRNA expression of Syndecan-2 (SDC2). In addition, our preliminary experiments evaluating SDC2 surface expression via flow cytometry suggest that both CD28 Low and CD86 Low populations have increased expression of SDC2. Additionally, in the RNA-seq analysis we also observed that CD28 Low and CD86 Low cells were also able to sustain mRNA expression of IRF4. This result was later confirmed via western blot, demonstrating CD28 Low and CD86 Low cells sustain protein expression of IRF4. Conclusions: Our results demonstrate that RPMI8226 cells that lack CD28 or CD86 expression have increased mRNA and protein expression of SDC2, a member the heparan sulfate proteoglycan family. Since SDC2 expression has also been associated with a number of cancers including colorectal, lung, and pancreatic cancers, these results provide insight to potential alternative pathways that contribute to myeloma survival independent of CD28 and CD86 signaling. We also confirmed that CD28- and CD86-depleted populations were able to maintain IRF4 mRNA/protein levels. This indicates myeloma cells have mechanisms to sustain IRF4 expression in addition to CD28 and CD86 signaling. These results are also consistent with previous findings illustrating importance of IRF4 in myeloma. Finally, we have developed CRISPR/Cas9-depleted CD28 and CD86 populations in additional HMCLs. We are currently in the process of isolating CD28 High/Low and CD86 High/Low populations for additional analysis and will present these upcoming results.

An Efficient Method to Prepare Barcoded cDNA Libraries from Plant Callus for Long-Read Sequencing.

Long-read sequencing methods allow a comprehensive analysis of transcriptomes in identifying full-length transcripts. This revolutionary method represents a considerable breakthrough for non-model species since it allows enhanced gene annotation and gene expression studies when compared to former sequencing methods. However, woody plant tissues are challenging to the successful preparation of cDNA libraries, thus, impairing further cutting-edge sequencing analyses. Here, a detailed protocol for preparing cDNA libraries suitable for high throughput RNA sequencing using Oxford Nanopore Technologies® is described. This method was used to prepare eight barcoded cDNA libraries from two Solanum betaceum cell lines: one with compact morphology and embryogenic competency (EC) and another with friable and non-embryogenic (NEC). The libraries were successfully sequenced, and data quality assessment showed high mean quality scores. Using this method, long-read sequencing will allow a comprehensive analysis of plant transcriptomes.

Selective ribosome profiling as a tool to study interactions of translating ribosomes in mammalian cells.

The processing, membrane targeting and folding of newly synthesized polypeptides is closely linked to their synthesis at the ribosome. A network of enzymes, chaperones and targeting factors engages ribosome-nascent chain complexes (RNCs) to support these maturation processes. Exploring the modes of action of this machinery is critical for our understanding of functional protein biogenesis. Selective ribosome profiling (SeRP) is a powerful method for interrogating co-translational interactions of maturation factors with RNCs. It provides proteome-wide information on the factor's nascent chain interactome, the timing of factor binding and release during the progress of translation of individual nascent chain species, and the mechanisms and features controlling factor engagement. SeRP is based on the combination of two ribosome profiling (RP) experiments performed on the same cell population. In one experiment the ribosome-protected mRNA footprints of all translating ribosomes of the cell are sequenced (total translatome), while the other experiment detects only the ribosome footprints of the subpopulation of ribosomes engaged by the factor of interest (selected translatome). The codon-specific ratio of ribosome footprint densities from selected over total translatome reports on the factor enrichment at specific nascent chains. In this chapter, we provide a detailed SeRP protocol for mammalian cells. The protocol includes instructions on cell growth and cell harvest, stabilization of factor-RNC interactions, nuclease digest and purification of (factor-engaged) monosomes, as well as preparation of cDNA libraries from ribosome footprint fragments and deep sequencing data analysis. Purification protocols of factor-engaged monosomes and experimental results are exemplified for the human ribosomal tunnel exit-binding factor Ebp1 and chaperone Hsp90, but the protocols are readily adaptable to other co-translationally acting mammalian factors.

RNA Sequencing Analysis of Saliva exRNA.

Next-generation sequencing (NGS) methodologies are rapidly developing. However, RNA Sequencing of saliva is challenging due to low abundance and integrity of extracellular RNA, as well as large amounts of bacterial RNAs that may be encountered in saliva. In addition, the literature about human salivary extracellular RNA is very scarce. Therefore, in our chapter, we present the most appropriate protocols for saliva collection, pre- and post-processing, including bioinformatic analysis of salivary RNA Sequencing data. However, the choice of the proper method for RNA extraction, cDNA library preparation, and computational pipeline can make a significant impact on the final quality of data and their interpretation.

ScRNA-seq for Microcephaly Research [II]: Preparation of Single-Cell cDNA Libraries.

Droplet-based single-cell RNA-seq (scRNA-seq) requires the addition of bar codes that mark the cell and transcript of origin. Cell-specific bar codes, typically added by cDNA elongation on beads, label each transcript derived from an individual cell. Transcript-specific bar codes serve as unique molecular identifiers and allow for the maintenance of transcript proportions after PCR-based amplification of cDNA. This chapter provides methods for generating bar-coded scRNA-seq libraries after droplet encapsulation.

CCRG-03. STUDY OF PIWI-INTERACTING RNAS IN GLIOBLASTOMA PATHOLOGY: A NEW LEVEL OF REGULATION OF GLIOBLASTOMA STEM CELLS?

Abstract BACKGROUND One of the putative mechanisms of early recurrence of GBM is the existence of glioblastoma stem cells (GSCs) that can resist therapy and subsequently establish new GBM foci. PIWI-interacting RNAs (piRNAs), responsible for maintaining genome stability, may then play a key role in their biology, particularly in germ and stem cells. A number of piRNA molecules are also promising biomarkers useful for prognosis and prediction of disease progression.Material and METHODS Glioblastoma stem cells were cultured in DMEM/F12 medium supplemented with bFGF and EGF growth factors. DMEM medium containing 10% fetal bovine serum (FBS) was used to culture differentiated cells. RNA was isolated from the selected paired primocultures and subsequently checked for quality by capillary electrophoresis. NEBNext Small RNA Library Prep Set was used for the preparation of cDNA libraries. Sequencing analysis was performed using the Next500/550 High Output v2 Kit for 75 cycles and the NextSeq500 sequencer. RESULTS Twenty paired primary cultures were prepared to verify the expression of CD133 and SOX2 in GSCs, the ability to establish tumors in immunodeficient mice, and the ability to differentiate. At this time, we finished sequencing of piRNA molecules and acquired data are bioinformatically analysed. Subsequently, we will perform analysis of selected piRNAs to be diagnostic and prognostic biomarkers on the GBM samples and non-tumor brain controls. All results will be presented at the conference. CONCLUSION Based on recent studies, it is evident that piRNA expression can have a significant impact on GBM biology. Their expression within GSCs may then provide a tool for identifying and targeting these cells and could be usable as a diagnostic and prognostic biomarker in GBM patients.The study was supported by the programme project of the Ministry of Health of the Czech Republic with reg. no. NV19-03-00501, NV19-03-00559, Conceptual development of research organization FNBr, 65269705

Enhanced transcriptome-wide RNA G-quadruplex sequencing for low RNA input samples with rG4-seq 2.0

BackgroundRNA G-quadruplexes (rG4s) are non-canonical structural motifs that have diverse functional and regulatory roles, for instance in transcription termination, alternative splicing, mRNA localization and stabilization, and translational process. We recently developed the RNA G-quadruplex structure sequencing (rG4-seq) technique and described rG4s in both eukaryotic and prokaryotic transcriptomes. However, rG4-seq suffers from a complicated gel purification step and limited PCR product yield, thus requiring a high amount of RNA input, which limits its applicability in more physiologically or clinically relevant studies often characterized by the limited availability of biological material and low RNA abundance. Here, we redesign and enhance the workflow of rG4-seq to address this issue.ResultsWe developed rG4-seq 2.0 by introducing a new ssDNA adapter containing deoxyuridine during library preparation to enhance library quality with no gel purification step, less PCR amplification cycles and higher yield of PCR products. We demonstrate that rG4-seq 2.0 produces high-quality cDNA libraries that support reliable and reproducible rG4 identification at varying RNA inputs, including RNA mounts as low as 10 ng. rG4-seq 2.0 also improved the rG4-seq calling outcome and nucleotide bias in rG4 detection persistent in rG4-seq 1.0. We further provide in vitro mapping of rG4 in the HEK293T cell line, and recommendations for assessing RNA input and sequencing depth for individual rG4 studies based on transcript abundance.ConclusionsrG4-seq 2.0 can improve the identification and study of rG4s in low abundance transcripts, and our findings can provide insights to optimize cDNA library preparation in other related methods.

Improved Nanopore full-length cDNA sequencing by PCR-suppression.

Full-length transcript sequencing remains a main goal of RNA sequencing. However, even the application of long-read sequencing technologies such as Oxford Nanopore Technologies still fail to yield full-length transcript sequencing for a significant portion of sequenced reads. Since these technologies can sequence reads that are far longer than the longest known processed transcripts, the lack of efficiency to obtain full-length transcripts from good quality RNAs stems from library preparation inefficiency rather than the presence of degraded RNA molecules. It has previously been shown that addition of inverted terminal repeats in cDNA during reverse transcription followed by single-primer PCR creates a PCR suppression effect that prevents amplification of short molecules thus enriching the library for longer transcripts. We adapted this method for Nanopore cDNA library preparation and show that not only is PCR efficiency increased but gene body coverage is dramatically improved. The results show that implementation of this simple strategy will result in better quality full-length RNA sequencing data and make full-length transcript sequencing possible for most of sequenced reads.

Abstract 6128: Establishment of spatial transcriptomics assay to study the mechanism of immune therapy against tumors

Abstract Background: Effective cancer immunotherapy is significantly affected by intratumoral heterogeneity, not only in different cell types and functions but also in the spatial pattern of immune cells within the tumor tissue. While most single-cell RNA sequencing technologies could enable the exploration of gene expression heterogeneity at the single-cell level, they cannot reveal spatial information within the tumor for us to map the whole transcriptome with morphological context. Spatial transcriptomics is an in situ capturing technique, which profiles gene expression at the RNA level, whilst preserving the spatial information of histological tissue sections. Thus we try to develop our spatial transcriptomics assay through a serial of validation experiments and to explore the mechanism of immune therapy against tumors. Material and Method: Firstly, the tumor was frozen and sections were cryosectioned at 10 μm thickness. Sections processing including H&E staining, tissue optimization, permeabilization, reverse transcription, and cDNA library preparation was carried out following the user guide of Visium Spatial Gene Expression Reagent Kits (10X Genomics). And the final cDNA libraries were sequenced using the Illumina Novaseq instrument. Using gene expression matrix and aligned H&E image processed by Cell Ranger and Loupe Browser with default parameters, we performed data QC, normalization, dimensionality reduction, and clustering by Seurat and cell components identification in tumor microenvironment based on a multistep approach. Differential gene expression and spatial heterogeneity were analyzed in each cluster. Results: Here we established a reliable system for spatial transcriptomics assay to elucidate the mechanism of anti-PD1 treatment in MC38 syngeneic colorectal tumor. Using this system, we constructed the gene expression spatial patterns of tumor cells and immune cells, including periphery and center of tumor cell, immune-cell enrichment area, and tumor cell surrounding the immune cell. Furthermore, we explored the cell-cell interactions in which the tumor cells directly interact with adjacent non-tumor cells, for example, macrophages. Using this system, we tried to investigate the mechanism of anti-PD-1 therapy against the tumors by comparing the spatial transcriptome profiles after the treatment with anti-PD-1 or isotype control in colorectal cancer models. Interestingly, changes in both gene-level and spatial levels were observed, and it will help reveal how the immune therapy may shake up the complex ecosystem. Summary: We established a reliable system for spatial transcriptomics analysis and explored the mechanism of anti-PD-1 treatment against the colorectal cancer by using this system. Citation Format: Panpan Wang, Huacheng He, Qiyao Zhang, Jingjing Wang, Dan Lu, Fei Duan, Qiangqiang Fan, Qingyang Gu, Qunsheng Ji. Establishment of spatial transcriptomics assay to study the mechanism of immune therapy against tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6128.

Abstract 908: Small RNA-seq analysis of PIWI-interacting RNAs in glioblastoma stem cells: Identification of new therapeutic targets in glioblastoma patients

Abstract Introduction: Glioblastoma (GBM) is the most common malignancy affecting the CNS. Despite radical therapy, recurrence of GBM is a frequent and relatively early event in the course of the disease. One of the putative mechanisms of early recurrence of GBM is the existence of glioblastoma stem cells (GSCs) that can resist therapy and subsequently establish new GBM foci. PIWI-interacting RNAs (piRNAs), responsible for maintaining genome stability, may play a key role not only in the biology of germ and stem cells but also GSCs since their dysregulated expression has been described in several cancers, including GBM. Thus, the identification of specific piRNAs in GSCs could allow the distinction of these cells from other GBM cells and their eventual targeting. Some piRNAs could also be promising biomarkers useful for prognosis and prediction of disease progression. Material and Methods: Native GBM tissues dissociated using the Papain Dissociation System were used for culturing of paired cell primary cultures. Cells were divided into two aliquots with different culture conditions. GSCs were cultured in DMEM/F12 medium supplemented with bFGF and EGF growth factors. DMEM medium containing 10% fetal bovine serum (FBS) was used to culture non-stem GBM cells. Subsequently, the presence of neural stem cell markers CD133 and SOX2, the ability of GSCs to establish tumors in immunodeficient mice, and the ability to differentiate into more mature cell types were analyzed. RNA isolated from the paired primary cultures was used for small-RNA sequencing (RNA-seq). QIAseq miRNA Library Kit was used for the preparation of cDNA libraries. Sequencing analysis was performed using the NextSeq 500/550 High Output v2 Kit (75 cycles) and the NextSeq 500 sequencer. Results: Twelve paired primary cultures were prepared to global piRNA expression profiling. Small RNA-seq bioinformatics analysis identified between 3.45 and 5.51% of unique mapped piRNA reads in all samples; GSCs expressing higher levels of piRNA molecules in comparison with paired GBM cells. Subsequently, we identified a set of significantly differentially expressed piRNAs in GSCs. Expression of selected piRNAs was then artificially regulated in primary GSCs in vitro, and the effect of these molecules on neurosphere formation, viability, and cell genome stability was analyzed. Finally, we also observed an association of piR-23231 with overall survival of GBM patients. All results will be presented at the AACR Annual Meeting 2022. Conclusion: Based on the results so far, piRNAs seem to play an important role in GSCs as well as GBM biology and their targeting may be a promising tool for therapy of GBM patients. The study was supported by the programme project of the Ministry of Health of the Czech Republic with reg. no. NV19-03-00501 and NV19-03-00559. Citation Format: Jiri Sana, Frantisek Siegl, Marek Vecera, Pavel Fadrus, Jaroslav Juracek, Petra Pokorna, Karolina Trachtova, Tomas Kazda, Ondrej Slaby. Small RNA-seq analysis of PIWI-interacting RNAs in glioblastoma stem cells: Identification of new therapeutic targets in glioblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 908.

Abstract 3140: Interleukin-34 regulates tumor immune microenvironment of renal cell carcinoma

Abstract Renal Cell Carcinoma (RCC) is a type of kidney cancer which derives from renal tubular epithelial cells and is responsible for approximately 90-95% of cases in adults. Incidence and prevalence of RCC are rising, and 5-year survival of metastatic disease is close to 10%. The therapy of RCC mainly targets the angiogenic and immunosuppressive tumor microenvironment (TME), but it is rarely curative and drug resistance is almost inevitable. Lack of validated biomarkers and scarce knowledge of the biological processes occurring during RCC progression are main reasons of therapy failure. Using an immunocompetent murine model of RCC, we identified Interleukin-34 (IL34) as potential biomarker of RCC progression, whose expression is associated with advanced tumor stage and reduced survival in RCC patients. In cancer, IL34 was demonstrated to regulate cancer cell proliferation and monocytes survival and differentiation into macrophages. Regarding to RCC, IL34 role in the TME still remains elusive and has never been described. To elucidate the function of IL34 in RCC biology, we undertook different approaches: Gene Ontology enrichment analysis of IL34 co-expressed genes, using the KIRC-TCGA database, which revealed that such genes are involved in different immune system related processes; orthotopic implantation of IL34 over-expressing murine renal cancer cells (i.e. RENCA cell line) in BALB/c mice, where we observed, both in primary tumors and lung metastases, an accumulation of tumor associated macrophages (TAM) expressing M2-type macrophage markers (e.g. Cd206 and Cd163). Subsequently, to deeply investigate how IL34 can finely tune the tumor immune microenvironment of RCC, we also employed an experiment of Single Nuclei Sequencing using murine samples over-expressing IL34 (or control empty vector). Briefly, RENCA cancer cells were implanted in BALB/c mice using two different injection modalities: orthotopic implantation in the kidney, to generate primary tumors; 2) tail vein injection, for lung metastases formation. After tumor or metastases formation, samples were snap-frozen in liquid nitrogen and processed for cDNA library preparation for next generation sequencing. Our project aims to elucidate how IL34 can be implicated in the regulation of TME in RCC and, in particular, of the phenotype of the accumulated TAM. As TAM are known to promote cancer progression by enhancing tumor angiogenesis and immunosuppression, the study of IL34 role in the TME can reveal novel paradigms in RCC biology, and may be fundamental to improve the current therapy (e.g. using drugs targeting IL34 activity). Citation Format: Andrea Emanuelli, Wilfried Souleyreau, Tiffanie Chouleur, Marie-Alix Derieppe, Andreas Bikfalvi. Interleukin-34 regulates tumor immune microenvironment of renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3140.

Spatial Transcriptomics Reveal Potential Sex Differences in Gene Expression of the Supraoptic Nucleus

The supraoptic nucleus (SON) of the hypothalamus contains magnocellular neurosecretory cells that play a key role in the regulation of fluid and electrolyte homeostasis. Although there are many well‐known sexually dimorphic regions of the hypothalamus, little is known about whether this is also true for the SON and whether there are sex differences in SON gene expression. Our study aims to address this knowledge gap by leveraging spatially‐resolved transcriptomics to better visualize gene expression profiles of cells in the SON of male and female rats and gain insight on their physiological functions without sacrificing morphological context. Visium Spatial Gene Expression (10x Genomics) was used to obtain spatially‐resolved gene expression data for the SON of adult male (n=3) and female (n=3) Sprague‐Dawley rats. Briefly, each brain was sectioned at 10μm thickness to collect coronal sections (~4x4mm) containing the SON and other brain structures. Each section was then mounted on the capture areas of Visium slides containing probes that bind mRNA. Next, the sections underwent the workflow as follows: 1) sample staining and imaging, 2) cDNA library preparation, 3) sequencing, and 4) analysis/data visualization. Data were analyzed using 10x Genomics’ Space Ranger and Loupe Browser applications to overlay spatial gene expression data with reference images. Results show that gene cluster analysis successfully differentiated myelinated fiber tracts from nuclei and identified several distinct neuronal populations in the coronal brain sections from both male and female rats. From the list of significant genes after performing differential expression analysis on the SON region using 10x Genomics’ Loup Browser, 74/157 genes were unique to the males, 228/311 genes were unique to the females, and 83 genes (e.g., Avp and Oxt) were common to both sexes. Preliminary Gene Ontology (GO) Enrichment and pathway analyses revealed GO terms and pathways related to: 1) homeostatic processes, regulation of peptide hormone secretion, and ion transport for the common genes; 2) ribosomes and translation for the significant genes unique to males; and 3) neurotransmitter release cycle and synaptic transmission for the significant genes unique to females, as some examples. These spatially‐resolved transcriptomic data suggest potential sex differences in SON gene expression that may be associated with processes and pathways important for osmoregulation and fluid balance. Future spatial transcriptomic studies will investigate changes in SON gene expression that contribute to sex differences in cellular mechanisms involved in body fluid homeostasis and possibly pathophysiology.

Microfluidic cell sorter sample preparation for genomic assays.

Single-cell RNA-Sequencing has led to many novel discoveries such as the detection of rare cell populations, microbial populations, and cancer mutations. The quality of single-cell transcriptomics relies heavily on sample preparation and cell sorting techniques that best preserve RNA quality while removing dead cells or debris prior to cDNA generation and library preparation. Magnetic bead cell enrichment is a simple process of cleaning up a sample but can only separate on a single-criterion. Droplet-based cell sorters, on the other hand, allows for higher purity of sorted cells gated on several fluorescent and scatter properties. The downside of traditional droplet-based sorters is their operational complexity, accessibility, and potential stress on cells due to their high-pressure pumps. The WOLF® Cell Sorter, and WOLF G2®, developed by NanoCellect Biomedical, are novel microfluidic-based cell sorters that use gentle sorting technology compatible with several RNA-sequencing platforms. The experiments highlighted here demonstrate how microfluidic sorting can be successfully used to remove debris and unwanted cells prior to genomic sample preparation resulting in more data per cell and improved library complexity.